The 2021 World Health Organization (WHO) Classification of Central Nervous System (CNS) classification formalized routine molecular profiling, and DNA-methylation studies have since delineated PLAG-family-altered CNS entities: PLAGL1 fusion-positive supratentorial neuroepithelial tumors (NET_PLAGL1) and embryonal tumors with PLAGL1/PLAGL2 amplification. PLAG1 fusions with diverse partners have been reported in CNS embryonal tumors, but have not been described in supratentorial neuroepithelial tumors to date. We describe two pediatric supratentorial ependymal tumors with novel PLAG1 fusions that do not match any 2021 WHO-defined entity or any PLAG-family-related entity recently reported in the literature. Case 1 (4-year-old boy) had a 7.6-cm left lateral ventricular mass with edema and heterogeneous enhancement; gross total resection was performed without adjuvant therapy (alive at 8 months). Histology showed diffusely low cellularity with small- to medium-sized round nuclei, minimal atypia, and focal calcifications; no ependymal rosettes, branching vessels, or clear-cell change. Tumor cells were positive for GFAP and H3K27me3, and negative for Desmin, EMA, OLIG2, L1CAM, NF-κB and H3K27M. The MIB-1 labeling index was ~ 5%. RNA-seq identified TNC::PLAG1 fusion; FISH showed PLAG1 rearrangement. DNA methylation clustered with spinal subependymoma, despite supratentorial location. Case 2 (4-year-old girl) had a 1.4 × 1.1 cm left parahippocampal lesion; resected without adjuvant therapy (alive at 4 months). Histology showed low-to-moderate cellularity with diffuse microcystic change, focal clear/vacuolated cells, and delicate branching vessels. Tumor cells were positive for GFAP, EMA, L1CAM, H3K27me3 and ATRX, and negative for EMA, Desmin, NF-κB, OLIG2, H3K27M, and CD34 (MIB-1 ~ 2%). RNA-seq identified TXNIP::PLAG1 fusion. Methylation did not reach a class threshold. Both cases ultimately warrant a final diagnosis of ependymal tumor, not elsewhere classified. To our knowledge, TNC::PLAG1 and TXNIP::PLAG1 are first-ever fusions reported in any tumor type. They also represent the first PLAG1 fusions identified in pediatric supratentorial ependymal tumors. These cases highlight the value of integrating histology, methylation profiling, and fusion detection, and suggest a new candidate supratentorial ependymal subtype with PLAG1 fusions, pending validation in larger series.

Spinal ependymal tumors are a diverse group of neoplasms encompassing four subtypes: spinal ependymoma (SP-EPN), spinal ependymoma, MYCN-amplification (SP-EPN-MYCN), spinal myxopapillary ependymoma (SP-MPE), and spinal subependymoma (SP-SE). However, the molecular differences among these subtypes remain largely unknown. Using an integrated multi-omics approach (whole-genome sequencing, RNA-seq, and mass spectrometry), we identified the distinct molecular characteristics of three subtypes except for SP-EPN-MYCN. In SP-EPN, abnormal enrichment of ciliary signaling, particularly involving the MKS complex, was evident. SP-MPE exhibited significant dysregulation of mitochondrial metabolism, reflecting a metabolic profile aligned with the Warburg effect. SP-SE tumors showed enhanced activity of immune-related pathways, including interferon signaling and extracellular vesicle dynamics, suggesting a distinct tumor microenvironment. This pilot study identifies candidate molecular markers in a single-center spinal ependymal tumor cohort. Not applicable.

Biologically and morphologically distinct from other ependymomas, myxopapillary ependymomas (MPEs) are rare, slow-growing glial tumors originating predominantly from the conus medullaris, cauda equina, or filum terminale. Gross total resection is the standard of care for primary MPE. Nevertheless, despite maximal resection, the risk of recurrence, usually within the neural axis, remains high. However, extra-neural metastases can also occur. Due to the rarity of the entity, there is a lack of consensus on the management of recurrences and extra-neural metastatic disease. We present a case report and literature review of this rare ependymal tumor. We describe a case of a male patient with MPE who developed multiple recurrences, treated with numerous surgical resections, radiotherapy, and salvage chemotherapy before eventually developing extra-neural metastatic disease to lungs, abdomen, and lymph nodes 37 years after initial diagnosis. A biopsy of an axillary lymph node confirmed histomorphology comparable to the primary histology. To our knowledge, there are <30 cases of extra-craniospinal metastatic MPE reported since 1955. Consequently, there is no major consensus on the treatment of extra-neural metastatic MPE. Case reports and series remain of utter importance to share experience and help customize management. From this angle, surgery, and radiotherapy are still used in the face of central nervous system recurrence and "limited" extra-neural spread, depending on the patterns of invasion. Chemotherapy has shown a modest effect so far; however, positive outcomes from targeted agents and immunotherapy (alone or combined) have been reported, which warrants further exploration.

Spinal ependymoma and myxopapillary ependymoma are the two most common spinal ependymal tumor types that feature distinct histological characteristics, genetic alterations and DNA methylation profiles. Their histological distinction may be difficult in individual cases and molecular diagnostic assessment, in particular DNA methylome profiling, may then be required to assign the correct diagnosis. Expression of the homeobox gene HOXB13 at the mRNA and protein levels has been reported as a frequent finding in myxopapillary ependymoma that may serve as a diagnostic marker for these tumors. Here, we evaluated the diagnostic role of HOXB13 immunostaining in 143 spinal neoplasms, comprising 54 histologically classified myxopapillary ependymomas, 46 histologically classified spinal ependymomas, and various other tumor types. Immunohistochemical results for HOXB13 protein were compared to molecular findings obtained by bead array-based DNA methylation and DNA copy number profiling, as well as next generation gene panel sequencing-based mutational analysis. Our findings indicate strong nuclear HOXB13 expression as a reliable diagnostic marker for molecularly confirmed myxopapillary ependymoma. Moreover, we provide evidence that differential HOXB13 protein expression is related to differential HOXB13-associated CpG site methylation in myxopapillary vs. spinal ependymomas, which can be assessed by targeted DNA methylation analysis. Taken together, immunohistochemistry for HOXB13 protein expression and targeted DNA methylation analysis of HOXB13 represent useful surrogate approaches that may substitute for DNA methylome profiling in routine diagnostics and facilitate precise classification of spinal ependymal tumors. In particular, strong nuclear HOXB13 immunoreactivity may serve as a novel diagnostic criterion for the classification of myxopapillary ependymoma.